Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
  • G01N33/56983—Viruses
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P25/00—Drugs for disorders of the nervous system
  • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P3/00—Drugs for disorders of the metabolism
  • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
  • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders
  • A61P37/08—Antiallergic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
  • A61P7/04—Antihaemorrhagics; Procoagulants; Haemostatic agents; Antifibrinolytic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
  • A61P7/06—Antianaemics
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
  • C12N2710/00011—Details
  • C12N2710/16011—Herpesviridae
  • C12N2710/16211—Lymphocryptovirus, e.g. human herpesvirus 4, Epstein-Barr Virus
  • C12N2710/16222—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
  • G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
  • G01N2333/01—DNA viruses
  • G01N2333/03—Herpetoviridae, e.g. pseudorabies virus
  • G01N2333/04—Varicella-zoster virus
  • G01N2333/045—Cytomegalovirus
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
  • G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
  • G01N2333/01—DNA viruses
  • G01N2333/03—Herpetoviridae, e.g. pseudorabies virus
  • G01N2333/05—Epstein-Barr virus
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2500/00—Screening for compounds of potential therapeutic value

Definitions

• the present invention relates to induction of immune tolerance, and in particular to the use of epitopes from infectious agents to induce immune tolerance to other antigens in individuals seropositive for those infectious agents .
• viruses escape immune recognition by disruption of antigen presentation pathways (Lorenzo et al., 2001) and epitope mutation (Erickson et al., 2001). Resistance is mediated by inhibiting apoptosis of virally infected cells. Counterattack comprises the killing of effector T cells (Mueller et al . , 2001). Epstein-Barr virus (EBV) has been shown to avoid detection and clearance by such mechanisms.
• EBV Epstein-Barr virus
• EBV is a human ⁇ -herpes virus carried as a latent infection by more than 90% of adults, replicating in B-cells and nasopharyngeal epithelial cells (Kieff, 1996) .
• the acute infection is controlled by a cyt ⁇ toxic response predominantly against EBV Nuclear Antigens 3A, 3B and 3C (Kieff, 1996) , but, in all cases, the virus enters a latent state in B-cells (Kieff, 1996) .
• LMP1 is part of a restricted panel of genes expressed during latency, and in several EBV-associated malignancies including Hodgkin's disease and nasopharyngeal carcinoma (Horikawa et al., 2000; Pallesen et al., 1991) .
• the protein acts as a constitutively activated tumor necrosis factor receptor, transforming cells through activation of molecules including nuclear factor kappa B and the anti-apoptotic protein A20 (Eliopoulos et al . , 1996, 1997; Huen et al . , 1995; Mosialos et al . , 1995; Young et al., 1998).
• Thl and Th2 cells which produce ⁇ -interferon ( ⁇ -IFN) and IL-4 respectively (Mossman and Coffman, 1989), but further, T regulatory (Tr) cell subpopulations with important roles in immunoregulation and tolerance have now been defined (Groux et al., 1997; Levings and Roncarolo, 2000; Roncarolo et al., 2000, 2001; Shevach et al., 1998; Stephens and Mason, 2000) .
• production of the Tri cytokine IL-10 can protect rodents against a number of immune- mediated diseases (Groux et al .
• Th3 cell secretion of transforming growth factor- ⁇ prevents spontaneous autoimmunity (Gorelik and Flavell, 2000) and mediates some forms of oral tolerance (Weiner, 1997) .
• Regulatory subpopulations characterized by CD25 expression have also been isolated from rodents (Seddon, and Mason, 2000; Shevach, 2000) , and more recently from human peripheral blood (Jonuleit, et al., 2001; Levings, et al., 2001), but in most reports the suppressive effects of these cells are non-specific and not dependent on cytokine production.
• Tr cells in controlling immune- mediated disease raises the prospect that viruses may exploit such regulation as a fourth major mechanism to evade immune clearance.
• LMPl cytotoxic T cells specific for LMPl are notable for their absence from infected individuals (Chapman et al.-, 2001) .
• Dukers et al . (2000) have recently suggested that LMPl contains peptide motifs which can exert direct immunosuppressive effects on peripheral blood mononuclear cells.
• infectious agents encode antigens comprising tolerogenic peptide sequences.
• a "tolerogenic" peptide sequence is meant a sequence which, when administered to cells of the immune system, along with a target antigen, tolerises the cells to that target antigen.
• populations of cells so treated retain their capacity to mount a response to other antigens in the absence of the tolerogenic sequence.
• the types of immune response which can be inhibited in this way include "defensive” immune responses against foreign antigens, including those administered therapeutically, as well as “pathogenic” immune responses as seen in autoimmune and allergenic diseases. These responses are often characterised by lymphocyte proliferation, expression of cytokines such as IL-4 or gamma-IFN, and induction of antibody response.
• cytokines such as IL-4 or gamma-IFN
• the cells to be tolerised will be from an individual who has previously been infected with the infectious agent from which the tolerogenic peptide is derived.
• these tolerogenic sequences can induce antigen-specific tolerance of mononuclear leukocytes to target antigens. This activity therefore contrasts with the non-specific immunosuppressive effects attributed to some virus-derived peptides, e.g. from retroviral envelope proteins (Haraguchi et al . , 1995) and EBV LMPl protein (Dukers et al . , 2000).
• the present inventors have shown that it is possible to identify such sequences by testing their ability to induce expression of IL- 10 in cells from a donor seropositive for the relevant infectious agent .
• the present invention provides a method for assessing the tolerogenicity of a test peptide sequence from an infectious agent, comprising the steps of:
• step (iii) correlating the result of step (ii) with the tolerogenicity of the sequence
• said cell population comprises mononuclear leukocytes from a donor previously infected by said infectious agent.
• T lymphocytes including CD4 + and CD8 + T lymphocytes
• B lymphocytes including CD4 + and CD8 + T lymphocytes
• NK natural killer cells
• mononuclear phagocytes monocytes and macrophages
• dendritic cells dendritic cells.
• the cell population comprises one or more of these types of cells.
• the cell population comprises at least T lymphocytes, preferably CD4 + lymphocytes, or at least one type of antigen presenting cell (APC) . More preferably, the cell population comprises at least T lymphocytes, preferably CD4 + lymphocytes, and at least one type of antigen presenting cell.
• An antigen presenting cell is any cell capable of presenting an antigen to a T lymphocyte in the context of an MHC class II molecule.
• APCs mononuclear leukocytes
• APCs dendritic cells
• the majority of nucleated cells are capable of acting as APCs under the appropriate conditions, e.g. when exposed to pro-inflammatory cytokines, and so the cell population may further comprise APCs which would not normally be regarded as mononuclear leukocytes.
• the cell population comprises mononuclear leukocytes derived from a donor previously infected by the relevant infectious agent.
• the donor may be seropositive for the infectious agent, i.e. have circulating antibodies specific for the infectious agent.
• the donor may not have circulating antibodies specific for the infectious agent, for example where insufficient time has elapsed since infection for detectable levels of antibodies to be raised, or where a substantial time has elapsed since infection and antibody levels have fallen below the threshold of detectability.
• seropositive will be used throughout this specification to refer to any individual previously infected by the relevant infectious agent, regardless of actual serological status, and the term “seronegative” should be construed accordingly, i.e as referring to an individual not previously infected by the infectious agent.
• the method may further comprise the steps of:
• step (ii) (b) comparing the results from step (ii) with the results from step (ii) (a) .
• step (iii) the individual results, or any combination of the results, from any of steps (ii) , (ii) (a) and (ii) (b) may be correlated with the tolerogenicity of the sequence.
• the greater the level of IL-10 expression induced in the seropositive population by the test peptide the more likely it is that the test peptide will be tolerogenic.
• IL-10 expression may be determined by any appropriate method. Suitable methods include specific detection of IL-10 protein, e.g. by ELISA (Deveraux et al., 2000), flow cytometry (Kreft et al . , 1992), non-competitive flow immunoassay (Kjellstrom et al., 2000), immunofluorescence
• the present invention further provides a method for assessing the tolerogenicity of a test peptide sequence from an infectious agent towards a target antigen, comprising the steps of:
• test composition (i) contacting a cell population with (a) said test peptide sequence and (b) a target antigen, to make a test composition, and
• said cell population comprises mononuclear leukocytes from a donor previously infected by said infectious agent.
• the cell population comprises at least one type of APC, which may or may not be a mononuclear leukocyte, as set out above.
• step (ii) the cell population will not be re-contacted with the test peptide in step (ii) .
• the method may further comprise the steps of:
• step (iv) correlating the result of step (iii) with the tolerogenicity of the test peptide sequence.
• the response of the cell population to the second challenge with the target antigen may be assessed by any method that enables a tolerised population to be distinguished from a non-tolerised population.
• a response of a non-tolerised population to a foreign antigen would be expected to include one or more of e.g. cell proliferation (typically lymphocyte proliferation), and expression of one or more cytokines (other than IL-10) such as IL- 4, IL-2, IL-12 and gamma-IFN.
• step (iii) may comprise the assessment of any one of these markers, or of any other suitable marker.
• the method may be performed in vivo or in vi tro .
• the method is performed in vi tro, e.g. in culture.
• the methods may be performed in any suitable model in vivo.
• step (ii) The purpose of re-contacting the cells with the target antigen in step (ii) is to confirm that the cells have been tolerised to the target antigen by the initial contact of step (i) .
• the test composition does not still contain appreciable amounts of the test peptide sequence, or of tolerogenic or immunosuppressive factors produced by the cells themselves, which might interfere with any reaction stimulated by the target antigen in step (ii) . Therefore, the method may include the step of allowing the cells to rest between steps (i) and (ii) , so that the activity of test peptide in the test composition is reduced, the cells are not still expressing tolerogenic factors which would interfere with any reaction in step (ii) , and the activity of residual tolerogenic factors produced by the cells during or in response to the initial tolerogenic challenge is reduced.
• IL-10 activity is used herein as a marker for tolerogenic factors generated by the PBMCs in step (i) .
• the method may additionally or alternatively comprise the step of washing the cells prior to step (ii) . Washing may be performed in conventional fashion. Typically, the cells will be rested after washing. ⁇ Fresh antigen presenting cells may be added before recontacting the cells with the target antigen in step (ii) .
• IL-10 may play an effector role in inducing tolerance, so reduction of IL-10 activity may also be achieved by specific neutralisation, e.g. addition of a neutralising factor to the cells, such as a neutralising anti-IL-10 antibody.
• the method may further comprise the step of contacting the cell population with a confirmatory antigen unrelated to the test sequence or the target antigen, to confirm that the cells retain their general reactive capability, even though their reactivity to the target antigen has been modified.
• any suitable antigen may be used as the target antigen or confirmatory antigen.
• These antigens may be primary antigens or recall antigens; that is to say, the cells in the assay may or may not have been exposed to them before.
• a typical primary antigen for assay use is KLH (keyhole limpet haemocyanin) , while for donors previously immunised with Bacille Calmette-Guerin (BCG) , purified protein derivative (PPD) from yc ⁇ jacterium tuberculosis is a suitable recall antigen.
• T cell mitogens such as Concanavalin A, which are generally regarded as relatively non-specific in their activation of T cells, can also be used as target or confirmatory antigens within the meaning of the present invention. It has been found that PBMCs can be rendered unresponsive to ConA, PPD and other antigens or stimuli by the techniques described herein, but still retain their ability to respond to other antigens.
• test peptide sequence which is capable of inducing IL-10 expression and/or antigen-specific tolerance in seropositive cells as described above may be regarded as a "tolerogenic peptide sequence" .
• a tolerogenic peptide sequence may therefore be used to modulate an immune response, either in vivo or in vitro, by administration to suitable seropositive mononuclear leukocytes along with a target antigen.
• This technique has a number of applications. For example, it may be used prophylactically, to prevent subsequent development of an inflammatory response to the target antigen, or to inhibit a pre-existing immune reaction to the target antigen.
• the present invention provides a method of tolerising a cell population to a target antigen, comprising contacting said cell population with
• said cell population comprises mononuclear leukocytes from a donor seropositive for said infectious agent.
• the cell population may be contacted with the tolerogenic peptide sequence and/or the target antigen directly.
• the cell population may be contacted with the tolerogenic peptide sequence and/or the target antigen indirectly, e.g. via APCs which would not normally be regarded as mononuclear leukocytes, as described above.
• APCs which would not normally be regarded as mononuclear leukocytes, as described above.
• a population of APCs may be contacted with the tolerogenic peptide sequence and/or the target antigen, and the cell population subsequently contacted with the population of APCs.
• the tolerogenic peptide and target antigen may be administered to the cell population, or to the population of APCs, either together or separately, and in any order. Thus it is not intended that the tolerogenic peptide sequence and target antigen must necessarily be administered simultaneously.
• a tolerogenic peptide sequence and a target antigen may be administered directly to a test subject or a subject to be treated, e.g. an individual who has previously been infected by the relevant infectious agent.
• the invention provides a method of treatment of a disease or condition mediated by an immune response against a target antigen, comprising administering a tolerogenic peptide sequence to an individual suffering from said condition or disease.
• the target antigen may also be administered, either with the tolerogenic peptide sequence or separately.
• a tolerogenic peptide sequence and a target antigen may be administered in vi tro to a cell population comprising mononuclear leukocytes from such an individual. These cells may then be introduced into a test subject, or a subject to be treated, e.g. the subject from whom they were originally derived.
• a tolerogenic peptide sequence and a target antigen may be administered in vi tro to a population of APCs.
• the population of APCs may then be contacted in vitro with a cell population comprising mononuclear leukocytes from an infected individual. That cell population, or a subset thereof e.g. some or all of the mononuclear leukocytes, may then be introduced into a test subject, or a subject to be treated, e.g. the subject from whom they were originally derived.
• the population of APCs may be administered to a test subject, or a subject to be treated, e.g. the subject from whom they were originally derived. In this case contact between the cell population and the tolerogenic peptide sequence and target antigen takes place in vivo, via the APCs.
• cells or tissues may be removed from a donor individual or individual to be treated, treated with the tolerogenic peptide sequence and a target antigen, and reintroduced to the donor.
• Suitable cells or tissues include particular type of antigen presenting cells, heterogeneous populations of cells, e.g. peripheral blood lymphocytes or subsets thereof, lymph nodes, etc.
• the cell population comprises at least T lymphocytes, preferably CD4 + T lymphocytes. More preferably, the cell population comprises at least T lymphocytes, preferably CD4 + T lymphocytes, and at least one type of APC. From the above description it can be seen that the cell population to be tolerised, may in some embodiments be considered to comprise cells in si tu in a test subject or subject to be treated.
• test subject or subject to be treated will typically be a mammal, and may be a human.
• a test subject may be a non-human mammal e.g. a rodent, rabbit, etc. and will typically be seropositive for the infectious agent.
• the test subject may be a non-human mammal with a severe combined immunodeficiency, comprising lymphocytes from a donor of the appropriate species seropositive for the infectious agent.
• severe combined immunodeficiency is meant a defect in lymphocyte maturation, so that the affected animal has low or undetectable levels of mature T and/or B lymphocytes.
• the mammal may be a rodent, for example a mouse or rat, such as the SCID mouse .
• the non-human mammal with the severe combined immunodeficiency is reconstituted with human lymphocytes seropositive for EBV, e.g.
• the target antigen may be a suitable test antigen as described above, or any antigen to which an inappropriate or undesirable immune response occurs or is likely to occur.
• the target antigen may be one implicated in a disease state, e.g. a self antigen implicated in an autoimmune condition, such as rheumatoid arthritis, or an allergic state such as hayfever.
• the target antigen maybe a protein, polypeptide or peptide, including an epitope of a protein, or any other suitable entity capable of provoking an immune reaction, such as polysaccharides, lipids, macromolecular complexes, cells, etc.
• auto-immune diseases in which specific antigens have been identified as potentially pathogenically significant include multiple sclerosis (myelin basic protein) , insulin-dependent diabetes mellitus (glutamic acid decarboxylase) , insulin-resistant diabetes mellitus (insulin receptor) , coeliac disease (gliadin) , bullous pemphigoid (collagen type XVII) , auto-immune haemolytic anaemia (Rh protein) , auto-immune thrombocytopenia (GpIIb/IIIa) , myaesthenia gravis (acetylcholine receptor) , Graves' disease (thyroid-stimulating hormone receptor), glomerulonephritis, such as Goodpasture' s disease (alpha3 (IV) CI collagen), and pernicious anaemia (intrinsic factor) .
• the target antigen may be an exogenous antigen which stimulates a response which also causes damage to host tissues. For example, acute rheumatic fever is caused by an antibody response to a Streptococcal antigen which cross-reacts with a cardiac muscle cell antigen.
• the target antigen may be one which provokes an atopic or allergic response, e.g. pollen (implicated in hayfever, e.g. Timothy Grass pollen), house dust mites (asthma), cosmetics, allergens administered via insect bites, nut allergens, or therapeutic products such as factor VIII, factor IX, blood group antigens, or monoclonal antibodies.
• the methods of ""the present invention may be used to suppress responses to allogeneic or xenogeneic cells or tissues, including primary and secondary mixed lymphocyte reactions, graft rejection, and graft versus host disease.
• a subject intended to receive a cellular transplant may be tolerised to antigens expressed by those cells.
• the transplant may be given in conjunction with tolerogenic peptide sequences as described herein, or nucleic acid encoding such peptide sequences, in order to tolerise the recipient to those cells.
• some or all of the cells to be transplanted may be engineered to express tolerogenic peptides.
• a cell to be transplanted may contain nucleic acid encoding a tolerogenic peptide sequence according to the present invention such that the cell is capable of expressing the tolerogenic peptide sequence.
• the optimum methodology will depend on the identity of the cells to be engineered.
• Antigen presenting cells e.g. dendritic cells, etc.
• Other cell types may be engineered so that they secrete the expressed sequence, in order that it can be presented by neighbouring APCs.
• the infectious agent from which the test or tolerogenic peptide sequence is derived, may be a virus.
• the virus is a herpesvirus encoding a viral IL-10 homologue, preferably EBV.
• the test or tolerogenic peptide sequence may be derived from an EBV protein, preferably EBV LMPl protein or LMP2 protein.
• EBV protein preferably EBV LMPl protein or LMP2 protein.
• test or tolerogenic peptide sequence may comprise one or more of the sequences pi to p75, or pi' to p96' . If desired, more than one test or tolerogenic peptide sequence may be administered, either simultaneously or sequentially.
• the present invention also provides a method of treating a disease mediated by an immune response against a target antigen, comprising the steps of administering (a) a tolerogenic peptide sequence from an infectious agent, and (b) the target antigen, to an individual seropositive for said infectious agent.
• Nucleic acids encoding test or tolerogenic peptides, and/or target antigens may be useful in all the methods of the present invention.
• a nucleic acid encoding that peptide and capable of supporting its expression may be used instead.
• DNA vaccination techniques are well known to the skilled person, as reviewed in Mor and Eliza (2001); Smith (2000); Schleef et al. (2000) andENSopoulos and Plebanski (2000) .
• administration of a nucleic acid sequence encoding that peptide sequence is also envisaged.
• contacting a cell population or population of antigen presenting cells with a peptide sequence is considered to encompass contacting the relevant cells with an appropriate nucleic acid.
• the present invention further provides a method of tolerising a cell population to a target antigen, comprising contacting said cell population with (a) a nucleic acid encoding said test peptide sequence, such that said test peptide sequence is expressed in said cell population, and
• said cell population comprises mononuclear leukocytes from a donor seropositive for said infectious agent.
• the target antigen is a protein, polypeptide or peptide
• a nucleic acid encoding the target antigen may be administered, so that the target antigen is expressed in said cell population.
• the target antigen need necessarily be a protein, polypeptide or peptide.
• nucleic acids in this way is considered to be applicable, mutatis mutandis, to any corresponding embodiment of the present invention in which administration of a peptide sequence is referred to.
• target antigens are protein or peptide
• nucleic acids having appropriate coding sequences may likewise be administered instead.
• cells may be contacted with peptides by contact with cells engineered to express the relevant peptides and either secrete them or present them in the context of MHC molecules .
• the present invention further provides a pharmaceutical composition
• a pharmaceutical composition comprising a tolerogenic peptide sequence from an infectious agent and a target antigen, in admixture with a pharmaceutically acceptable carrier.
• the tolerogenic peptide sequence is derived from EBV, e.g. LMPl or LMP2 as described above.
• the composition may comprise EBV LMPl protein, LMP2 protein, or a portion or fragment of either comprising a tolerogenic peptide sequence.
• the tolerogenic peptide sequence may comprise one or more of the LMPl peptide sequences PI to P75, and/or one or more of the LMP2 peptide sequences PI' to P96' described herein.
• the present invention further provides EBV LMPl and LMP2 proteins, and portions or fragments of either, for example, the peptide sequences PI to P75, or PI' to P96' comprising a tolerogenic peptide sequence, for use in a method of medical treatment.
• the present invention further provides EBV LMPl and LMP2 proteins, and portions or fragments thereof, for example, the peptide sequences PI to P75, or PI' to P96' comprising a tolerogenic peptide sequence, for use in the treatment of a condition mediated by an immune response directed against a target antigen.
• the present invention further provides EBV LMPl and LMP2 proteins, and portions or fragments thereof, for example, the LMPl peptide sequences PI to P75, and the LMP2 peptide sequences PI' to P96' comprising a tolerogenic peptide sequence, in the preparation of a medicament for the treatment of a condition mediated by an immune response directed against a target antigen.
• the medicament may further comprise the target antigen.
• the medicament will typically be formulated for administration to an individual previously infected by EBV.
• preferred peptides include P2, P4, P5, P6, P7, P8, P9, P10, P12, P13, P14, P15, P16, P17, P18, P20, P22, P23, P24, P25, P26, P27, P29, P30, P32, P34, P35, P39, P68, P71, P72.
• Particularly preferred peptides include P2, P4, P7, P14, P15, P18, P20, P22, P23, P24, and P32.
• the condition may be, for example, type I diabetes mellitus, coeliac disease, multiple sclerosis, rheumatoid arthritis, systemic lupus erythematosus, myaesthenia gravis, autoimmune haemolytic anaemia and thrombocytopenia, an atopic response e.g. hay fever or asthma, or other allergy, e.g. to an allergen such as a pharmaceutical product or nut allergens, or an alloimmune response, e.g. graft rejection, graft versus host disease, or a response to therapeutic products such as factor VIII, or monoclonal antibody therapy.
• the target antigens described above may be useful for treatment of these conditions.
• compositions and medicaments described herein may comprise nucleic acids encoding tolerogenic peptides and/or target antigens, as appropriate.
• composition comprising a cell for transplantation to a recipient, in admixture with a pharmaceutically acceptable carrier, said cell comprising nucleic acid encoding a tolerogenic peptide according to the present invention, such that said tolerogenic peptide sequence can be expressed by said cell.
• the nucleic acid preferably encodes an EBV protein, e.g. LMPl or LMP2, or a fragment thereof comprising a tolerogenic peptide sequence .
• the tolerogenic peptide sequence and the target antigen may be administered together or separately. In preferred embodiments, they are administered together. They may be provided as an admixture of separate components, as a complex, or covalently associated. Where the target antigen is a protein, the tolerogenic peptide sequence and target antigen may be provided as a fusion protein. Use of fusion proteins in this manner is applicable to all aspects of the invention.
• the cell population to be tolerised may comprise mononuclear leukocytes from any suitable species.
• the mononuclear leukocytes are mammalian, e.g. from livestock animals such as horses, cattle, etc., from domestic animals, such as dogs, cats, etc., or from humans.
• individuals to be treated by the methods of the present invention are preferably mammals, e.g. livestock animals such as horses, cattle, etc., domestic animals, such as dogs, cats, etc., and humans.
• test or tolerogenic peptide sequence as used herein, whether a test or tolerogenic peptide sequence, should not be taken to refer solely to a free peptide consisting essentially or exclusively of that sequence, although this is encompassed by the present invention. Without wishing to be bound by any particular theory, it is believed that the methods of the present invention are effective as long as the relevant sequence can be presented to T cells by antigen presenting cells within the population. Thus it is believed that the test or tolerogenic peptide sequence may constitute a T cell epitope, in that it is capable of being presented to T cells in the context of MHC molecules. Therefore the test or tolerogenic peptide sequence is preferably at least 6 amino acids in length, more preferably at least 8 amino acids in length.
• test or tolerogenic peptide sequence is capable of acting as an MHC class II-restricted T cell epitope.
• the chance that a peptide will be capable of acting as a T cell epitope can be determined by assessing its ability to bind to the antigen binding groove of MHC II molecules.
• Peptide motifs which bind particular MHC alleles are known, and computer programs are available which can identify such motifs within protein sequences (Sturniolo et al . (1999) ; Singh and Raghava (2001) ) .
• any T cell that responds to a given peptide can also respond in a similar way to other peptides containing substitutions in residues that are not critical for MHC binding or T cell receptor recognition, and even to certain peptides that are substituted in critical residues.
• Such immunological cross reactivity of peptides can be demonstrated by showing that a particular T cell is capable of responding to more than one peptide.
• Such experiments may be performed using T cell clones.
• Techniques for cloning T cells are well known in the art. Without wishing to be bound by any particular theory, T cells of Tri phenotype may be implicated in the mechanism underlying the methods described herein. Such T cells do not proliferate significantly in response to stimulation, and suppress proliferation of other cells, and so can be difficult to clone. However, suitable techniques are known - see e.g. MacDonald et al. (2002) .
• Tolerogenic peptides derived from infectious agents described herein, or identified using the methods herein, may be used to screen for immunologically cross reactive peptides which exert similar tolerogenic effects by stimulating a similar or overlapping T cell population. Such cross reactive peptides may be considered mimetics' of the infectious agent-derived tolerogenic peptides described herein.
• the present invention provides a method for assessing the tolerogenicity of a test peptide sequence, comprising the steps of:
• step (iv) correlating the result of step (iii) with the tolerogenicity of the test peptide sequence
• each said cell population comprises mononuclear leukocytes from a donor previously infected by an infectious agent
• said control peptide sequence is derived from said infectious agent.
• the control peptide sequence will have been previously shown to induce IL-10 expression in a cell population comprising mononuclear leukocytes from a donor previously infected by said infectious agent.
• the first and second cell populations are derived from the same donor individual.
• the first and second cell populations comprise T cell clones, preferably Tri T cell clones, shown to respond to the control peptide when appropriately presented by APCs .
• control peptide may comprise one or more of peptides PI to P75 and/or PI' to P96' described herein.
• compositions for use in the present invention may be tailored to a specific individual, by selecting peptides likely to bind to their MHC.
• compositions may be- designed to have a broader spectrum of activity, being applicable to a wider range of the population. This may be achieved by incorporating peptides capable of binding more than one MHC allele, and/or incorporating more than one test or tolerogenic peptide, each having different MHC specificity.
• These peptides may be provided in any appropriate form, e.g. as mixtures of separate peptides or as fusion proteins.
• test or tolerogenic peptide sequence may be administered as part of a longer peptide, polypeptide or protein.
• the sequence may be used in the context of the whole or part of the full length native protein.
• the peptide, polypeptide or protein may be administered in any appropriate form, e.g. in native or denatured conformation.
• any peptide, polypeptide or protein may comprise more than one tolerogenic peptide sequence within the meaning of the present invention.
• the EBV LMPl protein is believed to contain numerous individual peptide sequences capable of inducing tolerance to a target antigen in EBV- seropositive PBMCs, as described more fully in the Examples below.
• a peptide, polypeptide or protein comprising one or more tolerogenic epitopes may be utilised in admixture with target antigen, or may, for example, be provided covalently coupled with a target antigen, either by chemical linkage, or, where the target antigen is a protein, as a fusion protein.
• Peptides, polypeptides or proteins, including fusion proteins, for use in the methods or compositions of the present invention may be generated by any appropriate method, including chemical synthesis and recombinant expression.
• the present invention further provides individual peptides having any one of the sequences PI to P75 and PI' to P96' described herein.
• Preferred peptides have the sequences of P2, P4, P5, P6, P7, P8, P9, P10, P12, P13, P14, P15, P16, P17, P18, P20, P22, P23, P24, P25, P26, P27, P29, P30, P32, P34, P35, P39, P68, P71, P72.
• Particularly preferred peptides have sequences of P2, P4, P7, P14, P15, P18, P20, P22, P23, P24, and P32.
• the present invention provides isolated nucleic acid molecules encoding the test and tolerogenic sequences of the present invention.
• the open reading frame may be contiguous with an open reading frame encoding a desired target antigen, in order to encode a fusion protein as described above.
• the present invention provides an expression vector comprising the above tolerogenic sequence-encoding nucleic acid, operably linked to control sequences to direct its expression, as well as host cells transformed with the vectors.
• the present invention also includes a method of producing peptides of the preceding aspect, comprising culturing the host cells and isolating the tolerogenic peptides thus produced.
• the sequences can be incorporated into a vector having control sequences operably linked to the encoding nucleic acid to control its expression.
• the vectors may include other sequences such as promoters or enhancers to drive the expression of the inserted nucleic acid, nucleic acid sequences so that the tolerogenic sequence peptide is produced as a fusion, e.g. with one or more other such tolerogenic sequences, or with one or more target antigens, and/or nucleic acid encoding secretion signals so that the peptide produced in the host cell is secreted from the cell.
• Peptides/polypeptides/proteins can then be obtained by transforming the vectors into host cells in which the vector is functional, culturing the host cells so that the peptide is produced and recovering the peptide from the host cells or the surrounding medium.
• Prokaryotic and eukaryotic cells are used for this purpose in the art, including strains of E. coli , yeast, and eukaryotic cells such as COS or CHO cells.
• Suitable vectors can be chosen or constructed, containing appropriate regulatory sequences, including promoter sequences, terminator fragments, polyadenylation sequences, enhancer sequences, marker genes and other sequences as appropriate.
• Vectors may be plasmids, viral e.g. 'phage, or phagemid, as appropriate. For further details see, for example, "Molecular
• Cells and techniques may be selected such as to permit or enhance the folding and ⁇ or formation of disulphide bridges (see e.g.
• Peptides may be synthesized by any suitable method, such as by exclusively solid-phase techniques, by partial solid-phase techniques, by fragment condensation or by classical solution couplings.
• the peptide chain can be prepared by a series of coupling reactions in which the constituent amino acids are added to the growing peptide chain in the desired sequence. Briefly, N-alpha-protected amino acid anhydrides are prepared in crystallized form or prepared freshly in solution and used for successive amino acid addition at the N-terminus .
• the growing peptide (on a solid support) is acid treated to remove the N-alpha-protective group, washed several times to remove residual acid and to promote accessibility of the peptide terminus to the reaction medium.
• the peptide is then reacted with an activated N-protected amino acid symmetrical anhydride, and the solid support is washed.
• the amino acid addition reaction may be repeated for a total of two or three separate addition reactions, to increase the percent of growing peptide molecules which are reacted. Typically, 1-2 reaction cycles are used for the first twelve residue additions, and 2-3 reaction cycles for the remaining residues.
• Peptides are preferably prepared using the Merrifield solid phase synthesis, although other equivalent chemical syntheses known in the art can also be used as previously mentioned.
• Such solid-phase synthesis is commenced from the C-terminus of the peptide by coupling a protected alpha-amino acid to a suitable resin.
• a suitable resin can be prepared by attaching an alpha-amino- protected amino acid by an ester linkage to a chloromethylated resin or a hydroxymethyl resin, or by an amide bond to a benzhydrylamine (BHA) resin or paramethylbenzhydrylamine (MBHA) resin.
• BHA benzhydrylamine
• MBHA paramethylbenzhydrylamine
• the preparation of the hydroxymethyl resin is described by Bodansky et al., Chem. Ind. (London) 38, 1597-98 (1966).
• Chloromethylated resins are commercially available from Bio Rad Laboratories, Richmond, Calif, and from Lab. Systems, Inc. The preparation of such
• the C-terminal amino acid protected by Boc and by a side-chain protecting group, if appropriate, can be first coupled to a chloromethylated resin according to the procedure set forth in Chemistry Letters, K. Horiki et al. 165-168 (1978), using KF in DMF at about 60°C. for 24 hours with stirring, when a peptide having free acid at the C-terminus is to be synthesized.
• the success of the coupling reaction at each stage of the synthesis is preferably monitored by the ninhydrin reaction, as described by E. Kaiser et al., Anal. Biochem. 34, 595 (1970) .
• the coupling reactions can be performed automatically, as on a Beckman 990 automatic synthesizer, using a program such as that reported in Rivier et al . Biopolymers, 1978, 17, pp 1927-1938.
• the protected peptide resin is treated with liquid hydrofluoric acid to deblock and release the peptides from the support.
• the resin support used in the synthesis is selected to supply a C-terminal amide, after peptide cleavage from the resin. After removal of the hydrogen fluoride, the peptide is extracted into 1M acetic acid solution and lyophilized.
• the peptide can be isolated by an initial separation by gel filtration, to remove peptide dimers and higher molecular weight polymers, and also to remove undesired salts.
• Test and tolerogenic peptide sequences need not correspond exactly to the amino acid sequence of the agent infecting the host from which the PBMCs to be tolerised are derived. It is well known that proteins from wild type isolates of infectious agents often contain differences relative to the sequences of reference isolates of that agent. However, use of peptides synthesised according to reference sequences will typically provide the desired tolerogenic effects.
• test or tolerogenic sequence not from the agent infecting the host, but from a related agent, as long as the agents are sufficiently closely related for immunological cross-reactivity to occur, such that the desired tolerance is induced.
• test/tolerogenic sequences may exert their effects by being presented to T cells with a Tri phenotype (3) by antigen presenting cells. Therefore it may be desirable to introduce mutations into a tolerogenic peptide from a given infectious agent in order to enable it to bind to a broader range of MHC molecules, and thus be used to tolerise a larger proportion of a population towards target antigens.
• test or tolerogenic peptides may be used which differ from known or wild type sequences for the corresponding region of the infectious agent protein, as long as they retain sufficient tolerogenic capability. This can readily be determined by use of the methods of the present invention.
• Variant peptides can be produced by a mixture of conservative variation, i.e. substitution of one hydrophobic residue such as isoleucine, valine, leucine or methionine for another, or the substitution of one polar residue for another, such as arginine for lysine, glutamic for aspartic acid, or glutamine for asparagine.
• conservative variation i.e. substitution of one hydrophobic residue such as isoleucine, valine, leucine or methionine for another, or the substitution of one polar residue for another, such as arginine for lysine, glutamic for aspartic acid, or glutamine for asparagine.
• altering the primary structure of a polypeptide by a conservative substitution may not significantly alter the activity of that peptide because the side-chain of the amino acid which is inserted into the sequence may be able to form similar bonds and contacts as the side chain of the amino acid which has been substituted out.
• substitutions are in a region which is critical in determining peptide conformation.
• variants having non-conservative substitutions As is well known to those skilled in the art, substitutions to regions of a peptide which are not critical in determining its conformation may not greatly affect its activity because they do not greatly alter the peptide ' s three dimensional structure, and so may not affect the desired activity, e.g. MHC binding. In regions which are critical in determining the peptides conformation or activity such changes may confer advantageous properties on the polypeptide. Indeed, changes such as those described above may confer slightly advantageous properties on the peptide e.g. altered stability or specificity.
• variant peptides may be extended at the N- or C-termini, and the C-terminus may be amidated or have a free acid form.
• a peptide which is an amino acid sequence variant will generally share at least about 50%, 60%, 70%, 80%, 90% or more sequence identity with a wild type or reference sequence from the relevant infectious agent.
• sequence identity means strict amino acid identity between the sequences being compared.
• compositions of the present invention may comprise, in addition to the tolerogenic peptide sequences and optionally target antigens, a pharmaceutically acceptable excipient, carrier, buffer, stabiliser or other materials well known to those skilled in the art. Such materials should be non- toxic and should not interfere with the efficacy of the active ingredient.
• a pharmaceutically acceptable excipient e.g. oral, intravenous, cutaneous or subcutaneous, nasal, intramuscular, intraperitoneal routes .
• compositions for oral administration may be in tablet, capsule, powder or liquid form.
• a tablet may include a solid carrier such as gelatin or an adjuvant.
• Liquid pharmaceutical compositions generally include a liquid carrier such as water, petroleum, animal or vegetable oils, mineral oil or synthetic oil. Physiological saline solution, dextrose or other saccharide solution or glycols such as ethylene glycol, propylene glycol or polyethylene glycol may be included as required.
• compositions of the present invention comprise peptides as active agents
• they will typically be delivered by other routes, e.g. by intravenous, cutaneous or subcutaneous injection, or injection at the site of affliction, when the active ingredient will be in the form of a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability.
• isotonic vehicles such as Sodium Chloride Injection, Ringer's Injection, Lactated Ringer's Injection.
• Preservatives, stabilisers, buffers, antioxidants and/or other additives may be included, as required.
• the active agents e.g. tolerogenic peptide sequences and target antigens
• the peptides may be covalently conjugated to a water soluble polymer, such as a polylactide or biodegradable hydrogel derived from an amphipathic block copolymer, as described in U.S. Pat. No. 5,320,840.
• Collagen-based matrix implants such as described in U.S. Pat. No. 5,024,841, are also useful for sustained delivery of peptide therapeutics.
• a composition that includes a biodegradable polymer that is self-curing and that forms an implant in situ, after delivery in liquid form. Such a composition is described, for example in U.S. Pat. No. 5,278,202.
• the present invention provides a pharmaceutical composition
• a pharmaceutical composition comprising a tolerogenic peptide- encoding nucleic acid molecule and its use in methods of therapy or diagnosis.
• the composition may further comprise a target antigen- encoding nucleic acid molecule, which may be contiguous with the tolerogenic peptide-encoding nucleic acid molecule.
• the present invention provides a pharmaceutical composition
• a pharmaceutical composition comprising one or more tolerogenic peptide sequences as defined above and its use in methods of therapy or diagnosis.
• the composition may further comprise one or more target antigens.
• the present invention provides the above described tolerogenic peptide sequences and encoding nucleic acid molecules for use in the preparation of medicaments for therapy.
• Peptides may preferably be administered by transdermal iontophoresis.
• transdermal delivery This form of delivery can be effected according to methods known in the art.
• transdermal delivery involves the use of a transdermal "patch" which allows for slow delivery of compound to a selected skin region.
• patches are generally used to provide systemic delivery of compound. Examples of transdermal patch delivery systems are provided by U.S. Pat. No. 4,655,766 (fluid-imbibing osmotically driven system), and U.S. Pat. No. 5,004,610 (rate controlled transdermal delivery system) .
• transdermal delivery may preferably be carried out using iontophoretic methods, such as described in U.S. Pat. No. 5,032,109 (electrolytic transdermal delivery system), and in U.S. Pat. No. 5,314,502 (electrically powered iontophoretic delivery device) .
• permeation enhancing substances such as fat soluble substances (e.g., aliphatic carboxylic acids, aliphatic alcohols), or water soluble substances (e.g., alkane polyols such as ethylene glycol, 1,3- propanediol, glycerol, propylene glycol, and the like) .
• fat soluble substances e.g., aliphatic carboxylic acids, aliphatic alcohols
• water soluble substances e.g., alkane polyols such as ethylene glycol, 1,3- propanediol, glycerol, propylene glycol, and the like
• a "super water- absorbent resin" may be added to transdermal formulations to further enhance transdermal delivery.
• Such resins include, but are not limited to, polyacrylates, saponified vinyl acetate-acrylic acid ester copolymers, cross-linked polyvinyl alcohol-maleic anhydride copolymers, saponified polyacrylonitrile graft polymers, starch acrylic acid graft polymers, and the like.
• Such formulations may be provided as occluded dressings to the region of interest, or may be provided in one or more of the transdermal patch configurations described above.
• the modulators may be given orally or by nasal insufflation, according to methods known in the art.
• administration of peptides it may be desirable to incorporate such peptides into microcapsules suitable for oral or nasal delivery, according to methods known in the art.
• Administration is preferably in a "prophylactically effective amount” or a “therapeutically effective amount” (as the case may be, although prophylaxis may be considered therapy) , this being sufficient to show benefit to the individual.
• the actual amount administered, and rate and time-course of administration will depend on the nature and severity of what is being treated. Prescription of treatment, e.g.
• targeting therapies may be used to deliver the active agent more specifically to certain types of cell, by the use of targeting systems such as antibody or cell specific ligands. Targeting may be desirable for a variety of reasons; for example if the agent is unacceptably toxic, or if it would otherwise require too high a dosage, or if it would not otherwise be able to enter the target cells.
• these agents could be produced in the target cells by expression from an encoding gene introduced into the cells, e.g. in a viral vector.
• the vector could be targeted to the specific cells to be treated, or it could contain regulatory elements which are switched on more or less selectively by the target cells.
• a composition may be administered alone or in combination with other treatments, either simultaneously or sequentially dependent upon the condition to be treated.
• Figure 2 shows cytokine and proliferative responses of PBMC from EBV seropositive donors to a panel of LMPl peptides.
• cytokine ELISAs IL-10, IL-4, gamma-IFN
• proliferation assay The broken line on each chart shows the minimum level considered to be a positive response.
• Figure 4 shows flow cytometric analysis (23) of the phenotype of IL-10 synthesizing cells.
• cultured cells from two EBV seropositive donors (A+B and C+D) were analyzed for expression of CD4 and IL-10, with the % of double positive cells shown in the upper right quadrant of each panel.
• A+C were obtained from unstimulated cultures and B+D from cells stimulated with peptides P14 (aa 66-85) and P8 (aa 36-55) respectively (shown to induce IL-10 in these donors) .
• Figure 6 shows that IL-10 inducing LMPl peptides inhibit proliferative responses by PBMC from EBV seropositive donors against recall antigen (PPD) .
• the white bars show the proliferative and gamma-IFN responses obtained when PBMC from three EBV seropositive donors were stimulated with PPD, either alone, or together with IL-10 inducing LMPl peptides (P4,7,23,35 for Donor 1, P4 and 22 for Donor 2, and P4,18 and 31 for Donor 3), or control gamma-IFN inducing LMPl peptides (P28 for Donor 1, P56 for Donor 2 and P33 for Donor 3) .
• the black bars show the effects of adding a neutralizing anti-IL-10 antibody to duplicate cultures at 0.5 ⁇ g/ml.
• Figures 7 to 10 illustrate the specificity and persistence of LMP- 1-induced tolerance.
• panel (a) shows proliferative, ⁇ -IFN and IL-10 responses obtained when PBMC from a given donor were first stimulated in culture with the mitogen Con A, the recall antigen PPD, or the primary antigen KLH, alone or in combination with purified LMPl.
• stimuli were also administered in combination with an LMP-1-derived peptide. Cells were rested for seven days, washed to remove the antigens, and added to fresh irradiated autologous PBMC as a source of antigen presenting cells.
• Panel (b) shows the results of restimulating the control cells with each of the three stimuli.
• Panel (c) shows the results of restimulating the cells originally stimulated in the presence of LMP-1.
• Panel (d) shown only for Figures 7 and 9, shows the results of restimulating cells originally stimulated in the presence of LMP-1-derived peptide. Results are shown for three EBV-seropositive donors ( Figures 7, 8 and 9) and one seronegative donor ( Figure 10) .
• FIG 11 shows that antigen processing is required for the induction of IL-10 secretion by purified LMPl.
• IL-10 responses are shown when PBMC from an EBV-seropositive donor were stimulated with purified LMPl or IL-10-inducing LMPl peptides in the presence or absence of the processing inhibitor chloroquine. Shaded bars show control cultures lacking chloroquine; open bars show those with chloroquine .
• Figure 12 shows that responses to recall antigen (PPD) and allergen (house dust mite allergen - HDM) can be inhibited by both single LMPl peptides and combinations of peptides .
• Mix 1 contains LMPl peptides P4, P14, P18 and P23;
• Mix 2 contains LMPl peptides P4, P7, P14 and P32;
• Mix 3 contains LMPl peptides P7, P14, P18 and P23. Mixtures of peptides were administered to give a final concentration of 15 ⁇ g/ml of each peptide in the assay.
• Figure 13 shows the effects of LMPl peptides on the responses of PBMC from two donors (panels (a) and (b) ) to a selection of antigens and also in a mixed lymphocyte reaction (MLR) .
• Antigens included the autoantigen Rhesus D protein (RhD) , alpha3 (IV) NCI collagen, house dust mite allergen (HDM) and Timothy grass pollen (TG) , with PPD as a positive control.
• Peptide mixtures 1 to 3 are as in Figure 12.
• Figure 14 shows that tolerance induced to the allergens HDM and TG by LMPl is antigen specific and persists in the absence of LMPl peptide. Protocols were as described above for Figures 7 to 10. Panel (a) shows primary stimulation with antigen/allergen alone and with LMPl peptides; panel (b) shows the effect of restimulating the HDM and TG-treated cells with HDM, TG or PPD.
• Figure 15 shows that LMPl peptides can be used to inhibit the response to the autoantigen RhD in PBMC from a patient with autoimmune haemolytic anaemia.
• Figure 16 shows that LMPl peptides can be used to inhibit the response to allergens HDM and TG in PBMC from a patient with hay fever and asthma.
• Panel (a) shows primary stimulation with allergen or antigen alone and with LMPl peptides. Results from restimulation with HDM, TG and PPD are shown in panel (b) .
• LMPl induces high levels of IL-10 secretion by PBMC from EBV seropositive but not seronegative donors.
• PBMC from ten EBV seropositive donors were tested for the ability to respond to purified LMPl with either Th cytokine secretion or proliferation.
• IL-10 was the predominant cytokine measured, with no significant proliferative, gamma-IFN or IL-4 responses.
• Figure 1 shows representative results obtained from two seropositive donors. To confirm that the observed responses resulted from previous EBV infection, PBMC from two EBV seronegative donors were tested for responsiveness to the purified LMPl.
• PBMC from EBV seropositive donors respond strongly to multiple LMPl peptides by secreting IL-10.
• PBMC from four EBV seronegative donors were also screened with the panel of LMPl peptides. Reactivity was rare in this group, with totals of only nine IL-10, one ⁇ -IFN, one proliferative and no IL-4 responses. Moreover, all these responses were relatively weak (data not shown) .
• Cells responding to LMPl and LMPl peptides with IL-10 secretion are CD3 + CD4 + .
• LMPl and LMPl peptides suppress proliferative and gamma-IFN responses by stimulating IL-10 secreting Tri cells.
• Tri cells CD4 + T-cells biased towards IL-10 secretion
• Tri cells play an important role in immunoregulation (Groux et al., 1997; Levings and Roncarolo, 2000) and have been shown to inhibit inflammatory responses (Groux et al., 1997; Roncarolo and Levings, 2000) .
• LMPl and the peptide panel were predominantly mediated by Tri cells and sought to confirm that they were capable of mediating suppression.
• PBMCs from three seropositive donors and one seronegative donor were first stimulated in culture with the mitogen Con A, the recall antigen PPD, or the primary antigen KLH, alone or in combination with purified LMPl, and in two cases in combination with an LMP-1- derived peptide. Subsequently, cells were rested for seven days, washed to remove the antigens, and added to fresh irradiated autologous PBMC as a source of antigen presenting cells (Plebanski et al . , 1992) . Each group of cells was then restimulated with each stimulus. Results are shown in Figures 7 to 10.
• the Tri response to LMPl deviates T-cells recognizing a bystander antigen to adopt an anergic, IL-10 secreting phenotype.
• Such induction of anergy specific for other viral antigens that are co-expressed with LMPl may be important in the maintenance of EBV latency.
• IL-10 secretion from CD4 + T cells suggests that whole LMPl may induce such responses after the protein has been processed and presented as antigenic peptide fragments by the APC.
• molecules from other pathogens have been shown to induce IL-10, not after processing, but by direct interaction with innate pattern recognition receptors (McGuirk et al., 2002; Mills et al., 2002; Urban et al . , 2001).
• PBMC cultures with chloroquine-treated or control APC, were stimulated with purified LMPl or IL-10-inducing LMPl peptides.
• the results show that inhibition of Ag processing prevents IL-10 secretion induced by purified LMPl, but not by the LMPl peptides .
• Both Thl and Th2 responses can be inhibited by single LMPl peptides and combinations of peptides .
• the effects of selected LMPl peptides and combinations of peptides on responses of seropositive PBMCs to PPD and house dust mite (HDM) allergen were assessed.
• PPD was chosen as it gives a response representative of a Thl-type response
• HDM was chosen to give a representative pathogenic IL-4-dominated allergic-type Th2 response.
• Peptide mixtures were chosen to minimise the chances of any given dqnor failing to produce an IL-10 response when stimulated with the mixture.
• Figure 12 shows results from one representative donor. As expected, administration of PPD alone provokes proliferation accompanied by IFN-gamma secretion, while HDM alone provokes proliferation and IL-4 secretion. In both cases, though, these reactions were suppressed by all three mixtures of peptides .
• LMPl peptides suppress responses of normal individuals to auto- and alloantigens and in mixed lymphocyte reations .
• Figure 13 shows responses of PBMCs from two normal individuals to antigens implicated in auto- and alloimmune responses were investigated. These donors gave the expected reactions to PPD, ConA and KLH (although unusually in this assay stimulation with KLH alone resulted in significant amounts of IL-10 production) which were suppressed by LMPl peptide mixtures.
• RhD The Rhesus D protein
• RhD The Rhesus D protein
• Alpha3 (IV) NCI (a3) is a collagen which is the target in Goodpasture' s disease.
• the Thl responses to both of these antigens of pathogenic significance was also inhibited in these individuals and deviated to IL-10 production.
• PBMCs use of allogeneic PBMCs as stimulators of a mixed lymphocyte reaction (MLR) with donor cells resulted in massive proliferation and appreciable gamma-interferon secretion. Yet again, the LMPl peptide mixes were able to profoundly inhibit even these very strong responses. In panel (b) , there can be seen to be IL-10 secretion instead. in panel (a) , there is slightly less strong inhibition and no significant IL-10 production (it is possible that this is due to an error in sampling the wrong time point) . These MLRs serve as a model for mis-matched HLA in transplant situations.
• MLR mixed lymphocyte reaction
• Th2 responses to allergens is antigen-specific and persistent PBMCs from a normal donor were stimulated with the allergens HDM and TG alone and in combination with peptide mixtures 1 to 3. Various other antigens were used as controls. Responses are shown in Figure 14, panel (a) . Panel (b) shows the results of restimulation with HDM, TG and PPD. In all cases, responses to the primary stimulus were suppressed but the cells retained the capacity to respond normally to other antigens. Thus suppression of IL-4-driven Th2 responses to allergens is antigen-specific and persists in the absence of LMPl peptide.
• LMPl peptides can inhibit Thl-type autoimmune responses in cells from affected individuals
• Epstein-Barr virus rather than avoiding detection, instead subverts the immune response by stimulating regulatory CD4 + T-cells that secrete the inhibitory cytokine interleukin-10 (IL-10) .
• regulatory T- cells are well recognized (1-3) but not known to have a role in viral persistence (4-6) .
• LMPl latent membrane protein 1
• IL-10 responses characteristic of T regulatory 1 (Tri) cells, coincided with inhibition of T-cell proliferation and ⁇ -interferon ( ⁇ -IFN) secretion induced by both mitogen and recall antigen.
• ⁇ -IFN ⁇ -interferon
• a possible explanation for the propensity of LMPl to elicit IL-10 production by Tri cells is that the establishment and maintenance of a suppressive response to LMPl results from IL-10 Conditioning' .
• Activation of CD4 + T-cells in the presence of IL- 10 leads to the generation of Tri cells (Groux et al., 1997) .
• EBV is one of the viruses that encodes a homologue of this cytokine, viral IL-10 (vIL-10) (Hsu et al, 1990) , which is expressed during lytic cycle infection (Hayes et al., 1999).
• Blood samples were obtained by venepuncture from a group of healthy volunteers. The donors were classified as EBV seropositive or seronegative by an ELISA for serum anti-EBNAl IgG, with negative results confirmed by immunofluorescence staining for IgG and IgM anti-viral capsid antibody.
• Blood samples were also obtained by venepuncture from a male patient with allergic rhinitis, a female patient with atopic asthma, and a male patient with warm-type idiopathic autoimmune haemolytic anaemia.
• LMPl was immunopurified from lysed EBV transformed B cells using the anti-LMPl antibody CS1-4 (Novocastra Laboratories) conjugated to anti-mouse IgG x coated magnetic beads (Biomag, PerSeptive Biosystems) .
• Peptide sequences are as follows:
• control antigen mycobacterial PPD Statens Seruminstitut
• T-cell mitogen Con A Sigma
• KLH primary antigen keyhole limpet hemocyanin
• RhD protein was prepared as described in Hall, A.M., Ward, F.J., Vickers, M.A., Stott, L-M., Urbaniak, S.J. & Barker, R.N. (2002). Interleukin-10 mediated regulatory T-cell responses to epitopes on a human red blood cell autoantigen. Blood 100:4529-4536. RhD " protein was added to cultures at an estimated concentration of 5 ⁇ g/ml. Timothy grass pollen extract and house dust mite ( Dermatophagoides pteronyssinus) extract (both International
• PBMC peripheral blood mononuclear cells
• Th cytokines ⁇ -IFN, IL-4 and IL-10 were assessed in duplicate lOO ⁇ l aliquots taken five days after stimulation of the cultures, using a sensitive cellular ELISA (Devereux et al . , 2000). Cytokine responses over twice the production in unstimulated cultures were considered positive (Devereux et al., 2000).
• the phenotypes of cultured cells that proliferate or secrete cytokine in response to antigen were determined by flow cytometry. Aliquots of PBMC were taken from responding cultures and stained with anti-CD3 phycoerythrin-Texas Red ® -x and anti-CD4 fluorescein isothiocyanate, with anti-CD25 phycoerythrin-cyanin 5.1 (all Beckman Coulter) in some experiments. Activated cells in proliferating cultures were identified using anti-CD71- phycoerythrin (PE) or anti-CD69-PE (both Beckman Coulter) .
• PE anti-CD71- phycoerythrin
• anti-CD69-PE both Beckman Coulter
• the cells were then pulsed with LMPl or IL-10- inducing LMPl peptide for 3 h, before being washed three times. They were then used as a source of APC in proliferation and cytokine assays at 10 6 cells/ml.
• IL-10 acts on the antigen- presenting cell to inhibit cytokine production by Thl cells . J Immunol. 146, 3444-3451.
• Epstein-Barr virus latent membrane protein 1 blocks p53-mediated apoptosis through the induction of the A20 gene. J. Virol. 70, 8653-8659.
• T-regulatory 1 cells a novel subset of CD4 T cells with immunoregulatory properties. J Allergy Clin Immunol. 106, S109-112.
• Plebanski, M. Saunders, M. , Burtles, S.S., Crowe, S. and Hooper,
• CD25 is a marker for CD4+ thymocytes that prevent autoimmune diabetes in rats, but peripheral T cells with this function are found in both CD25+ and CD25- subpopulations . J. Immunol. 165, 3105-3110.
• CD4+CD25+ immune regulatory cells are required for induction of tolerance to alloantigen via costimulatory blockade. J. Exp. Med. 193, 1311- 1318.

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